BMC Genomics
BioMed Central
Open Access
Research article
The mitochondrial genome of the 'twisted-wing parasite' Mengenilla
australiensis (Insecta, Strepsiptera): a comparative study
Dino P McMahon*, Alexander Hayward and Jeyaraney Kathirithamby
Address: Department of Zoology, University of Oxford, The Tinbergen Building, South Parks Road, Oxford, OX1 3PS, UK
Email: Dino P McMahon* - [email protected]; Alexander Hayward - [email protected];
Jeyaraney Kathirithamby - [email protected]
* Corresponding author
Published: 14 December 2009
BMC Genomics 2009, 10:603
doi:10.1186/1471-2164-10-603
Received: 15 July 2009
Accepted: 14 December 2009
This article is available from: http://www.biomedcentral.com/1471-2164/10/603
© 2009 McMahon et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Strepsiptera are an unusual group of sexually dimorphic, entomophagous
parasitoids whose evolutionary origins remain elusive. The lineage leading to Mengenilla australiensis
(Family Mengenillidae) is the sister group to all remaining extant strepsipterans. It is unique in that
members of this family have retained a less derived condition, where females are free-living from
pupation onwards, and are structurally much less simplified. We sequenced almost the entire
mitochondrial genome of M. australiensis as an important comparative data point to the already
available genome of its distant relative Xenos vesparum (Family Xenidae). This study represents the
first in-depth comparative mitochondrial genomic analysis of Strepsiptera.
Results: The partial genome of M. australiensis is presented as a 13421 bp fragment, across which
all 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes and 18 transfer RNA (tRNA)
sequences are identified. Two tRNA translocations disrupt an otherwise ancestral insect
mitochondrial genome order. A+T content is measured at 84.3%, C-content is also very skewed.
Compared with M. australiensis, codon bias in X. vesparum is more balanced. Interestingly, the size
of the protein coding genome is truncated in both strepsipterans, especially in X. vesparum which,
uniquely, has 4.3% fewer amino acids than the average holometabolan complement. A revised
assessment of mitochondrial rRNA secondary structure based on comparative structural
considerations is presented for M. australiensis and X. vesparum.
Conclusions: The mitochondrial genome of X. vesparum has undergone a series of alterations
which are probably related to an extremely derived lifestyle. Although M. australiensis shares some
of these attributes; it has retained greater signal from the hypothetical most recent common
ancestor (MRCA) of Strepsiptera, inviting the possibility that a shift in the mitochondrial selective
environment might be related to the specialization accompanying the evolution of a small,
morphologically simplified completely host-dependent lifestyle. These results provide useful
insights into the nature of the evolutionary transitions that accompanied the emergence of
Strepsiptera, but we emphasize the need for adequate sampling across the order in future
investigations concerning the extraordinary developmental and evolutionary origins of this group.
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Background
Strepsiptera are an unusual group of obligate endoparasitoid insects [1]. They occur as a small (approx. 600 spp.)
monophyletic insect order with uncertain evolutionary
origins, inclusive of any clear understanding over the
group's nearest extant relative. Strepsiptera parasitize 7
orders of insects, including silverfish (Thysanura); cockroaches (Blattaria); mantids (Mantodea); crickets and
grasshoppers (Orthoptera); bugs (Hemiptera); wasps,
ants and bees (Hymenoptera) and flies (Diptera). Understandably, the very uniqueness of Strepsiptera makes their
placement within insects using morphological taxonomic
methods a contentious task. Strepsiptera have been placed
as the sister-group to myriad different insect groups, from
beetles to true flies, even being placed outside of Holometabola: each hypothesis being founded on one or two
'key' characteristics that at one time or another have come
under question [2-15]. Confident assertions of classification have especially been restricted because intermediate
forms have largely gone extinct and are unrecorded in the
fossil record (see [16] for a notable exception). Simplification of gross morphology during strepsipteran evolutionary specialization can also be viewed as a significant
component of this problem: observable morphological
variation is low and unevenly distributed between
extremely dimorphic sexes.
Females are especially strongly simplified (lacking in most
typical adult characters such as wings, legs or mouthparts), and in most strepsipteran species, they remain in
the living host until the end of the reproductive cycle.
Conversely, males metamorphose in a typical holometabolan fashion; developing wings and a usual suite of adult
insect characters (such as antennae, mouthparts and compound eyes) only to leave the host immediately in search
of a mate; usually in the form of a female-containing host
[17-19]. Males deliver sperm through a brood canal opening in the cephalothorax (the modified "head") of the
female, who as a reproductive adult is only partially
exposed to the environment, as an extrusion between the
tergites or sternites of living hosts. After fertilization,
females are capable of producing many hundreds of thousands of active 1st instar larvae who emerge from the
cephalothorax.
Members of the strepsipteran lineage, Mengenillidae, are
the sister-group to Stylopidia, a clade that includes all
other extant Strepsiptera [[20], unpublished data]. This
family represents a transitional phase in the evolutionary
specialization of Strepsiptera, whereby both sexes leave
the host before pupation, and females do not reproduce
or release progeny in the unusual fashion outlined above.
The level of simplification in free living mengenillid
females is much less extreme than in Stylopidia (e.g. X.
vesparum); legs, mouthparts and compound eyes are all
http://www.biomedcentral.com/1471-2164/10/603
still present, although strongly reduced. The invention of
a completely endoparasitic female was probably one of
the most important novelties leading to the radiation of
this unique group [21]. Evolutionary studies attempting
to unravel the developmental and ecological phenomena
that make Strepsiptera so biologically interesting therefore require that species from before this important transition occupy a central role in research.
Insect mitochondrial genomes provide a useful medium
to deepen and connect comprehension of microevolutionary forces of populations, like neutral drift and selective sweeps, to macroevolutionary events affecting species
and/or deeper levels of divergence. The abundance of
mitochondria in most metazoan cell and tissue types
makes mtDNA an easily obtainable, universally plentiful
marker, where a lack of introns or duplicate genes and
non-coding variable spacer regions make amplification of
mtDNA relatively uncomplicated [22] (although pseudogenes co-opted by the nucleus from the mitochondrial
genome can create separate difficulties [23-26]). The nearcomplete sequence of the mt genome of Xenos vesparum is
available, and as a member of the more specialized clade
Stylopidia, it is important to place it into context. The
mitochondrial genome of Mengenilla australiensis (Family
Mengenillidae) was therefore chosen for sequencing.
Genome arrangement, nucleotide content, codon usage
and the secondary structure of ribosomal RNA genes are
each comparatively assessed between two distant Strepsiptera, and more widely across Holometabola (a major
division of insects defined by the process of complete metamorphosis, including the 4 very species-rich orders; Lepidoptera, Diptera, Hymenoptera, Coleoptera and 6
smaller orders).
Results and discussion
Genome composition
The near-entire mt genome fragment of M. australiensis is
presented as a 13421 bp sequence, across which 13 protein-coding genes (PCGs), a large (16S) and a small (12S)
subunit rRNA gene (rrnL and rrnS respectively) and 18
tRNA sequences can be identified (Figure 1). PCGs are in
expected positions, but two tRNA translocations disrupt
an otherwise ancestral genome arrangement. Serine1 (S1:
AGN) moves to a position between Alanine (A) and
Arginine (R), whereas Valine (V) is either lost, or transferred to a position in a region that spans the flanking
region, potentially anywhere between tRNA-Ile and nad2.
All start codons across the PCGs (nad2 is only partial)
begin with the typical M- or I- residue [27], and end in
TAA, TAG, TA, or T. The control region itself, including up
to 4 flanking tRNA genes could not be amplified. A variety
of approaches were explored, none of which were successful in spanning the presumed A+T region. Similar difficulties were encountered during the amplification of the mt
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Figure 1 schematic of the mt genome of M. australiensis
Summary
Summary schematic of the mt genome of M. australiensis. Genes transcribed on the leading (α) strand are given in
blue (PCGs) or light blue (tRNAs) and run clockwise. Genes of the opposite polarity are given in red (PCGs) or orange
(tRNAs). Abbreviations are described in the text.
genome of X. vesparum [28]. In our study, genome-specific
primers designed to span the A+T region also performed
well when reverse compliment versions were implemented. It is hypothesized that the region is unusually
long and/or too problematic for even advanced Taq
polymerases to amplify, as a result of extreme repetitiveness or secondary structural folding issues (or both).
Figure 2 compares the genome organization of M. australiensis and X. vesparum against the inferred ancestral
arrangement [29,30]; the former having just two gene
order rearrangements, compared to 4 in the latter. In the
context of variation across a dataset containing 68 mitochondrial genomes taken from 6 major holometabolan
groups compiled from Genbank (see methods), this
would be consistent with a mostly ancestral arrangement
being retained between the origins of the major holometabolan radiations, since every order contains representative ancestral (or near-ancestral as in M. australiensis)
genome arrangements. Within certain orders, especially
Hymenoptera (and certain hemimetabolous groups not
discussed here [31]), lineages have undergone significant
independent modifications (see [32] for a full review of
hymenopteran mt genome evolution). Some synapomorphic alterations to this ancestral arrangement also exist in
certain orders. These include a tRNA arrangement shift
from IQM to MIQ across Lepidoptera [33] (outside of the
fragment shown in Figure 2), and a likely shift from WCY
to CWY in Neuroptera - a modification that is not shared
by the other neuropterid orders [34]. But largely, the 68
genomes corroborate a hypothesis positing that the
holometabolan orders emerged during a (relatively) brief
period of ancient rapid radiation. Or in the context of
phylogenetic tree shape, as an initial phase of short internal nodes, during which there would be insufficient time
for changes in mitochondrial genome organization to
become fixed between orders, followed by a longer period
of external node expansion [35] in which enough time
would pass for modifications to emerge independently
along multiple branches within and between orders. With
the inclusion of M. australiensis, whose genome does not
share any of the alterations of X. vesparum, and is more
representative of an ancestral insect arrangement, this also
appears consistent for Strepsiptera: although greater sampling is required for a general picture of mt genome structural evolution to emerge.
The lengths of PCGs are truncated in both strepsipteran
species, and especially so in X. vesparum. Across the 68
holometabolan dataset, M. australiensis has a reduction in
mean content of 6.2 amino acids per gene. In X. vesparum,
this reaches 11.8 amino acid deletions per gene. X.
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Figurecomparison
2
of partial mt genome structure in M. australiensis and X. vesparum
Linear comparison of partial mt genome structure in M. australiensis and X. vesparum. Elements in red denote
deviations from the ancestral holometabolan arrangement [29,30]. Readers are referred to the primary research for a complete catalogue of genome rearrangements in Holometabola: Hymenoptera [32]; Coleoptera [88-93]; Neuropterida [34,94,95],
a shift from WCY to CWY is probably synapomorphic for Neuroptera but not for other neuropterid orders; Lepidoptera
[33,75-81] are characterized by a synapomorphic tRNA rearrangement MIQ, from IQM, 5' of nad2 (not shown); Diptera [6074].
vesparum has the shortest nad2, cox1, cox2, atp8, atp6, cox3,
nad3 and nad4 gene (Additional file 1), representing a
4.3% loss in amino acids from the average holometabolan complement. Its total coding genome is shorter by 154
amino acids, the next shortest is M. australiensis (81) followed by Bombus ignitus (67) and M. bicolor (45) (Figure
3). Given that substantial loss of gene content might be
expected to severely compromise gene functionality and
efficiency, it would be of considerable interest to investigate whether this kind of genomic streamlining is related
in any way to the peculiar lifestyle of small endoparasitoid
insects. In particular, does bottlenecking in strepsipteran
populations lead to slightly deleterious mutations, like
codon deletions, being fixed through random drift? Strepsipteran metapopulations could be experiencing the
extreme extinction-recolonization population dynamics
required for low effective population sizes to become
influential, but there are no a priori reasons to suspect
these should be so different from other hymenopteran
host-parasitoid systems that depend on similar insect host
groups (e.g. Evania appendigaster, Venturia canescens; both
sampled in this study). Alternatively, bottlenecking could
result from the population dynamics of mitochondria
themselves, through very low numbers of these organelles
being passed via the germ line of strepsipteran eggs, which
are known to be extremely small [36].
Analyses to date do not reveal general patterns of mitochondrial genome evolution across holometabolan parasitic lineages [37,38], but these have concentrated largely
on the comparative analysis of genome organization
(gene order/orientation) itself. Within orders like
Hymenoptera, although a correlation between the extent
of mt genome modification and parasitism appears to be
lacking [39,40] (with an additional implication that the
position of mt genes might largely be neutral [32]), the
truncation of PCGs as presented in this report, probably
represents a separate issue. Determining the extent to
which PCG truncation occurs within Strepsiptera must be
a target for future investigations. Unfortunately, direct
measures of insect mitochondrial effective population
size have not yet been investigated.
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Figure
Comparison
3 of the total mt coding genome size (in amino acids) across Holometabola
Comparison of the total mt coding genome size (in amino acids) across Holometabola. Black: Thysanura; Green:
Diptera; Orange: Lepidoptera; Red: Hymenoptera; Blue: Coleoptera; Turquoise: Neuropterida; Burgundy: Strepsiptera. Each
bar represents one species' genome. The superimposed line indicates the mean coding genome size across all 68 taxa.
Overall nucleotide composition is typically A+T rich in M.
australiensis (84.3%; 6th highest amongst holometabolan
insects), and like in X. vesparum, C-skew is high (+0.27). A
scatter of the variation in skew and A+T% across the 68
holometabolan genomes (plus 3 thysanuran genomes) is
shown in Figures 4A and 4B. Notably, in Figure 4B, of the
most C-skewed and G+C% poor genomes (occupying the
lower right quarter of this graph), 9 (of 12) data points are
hymenopteran, 2 (of 2) are strepsipteran and 1 (of 12) is
lepidopteran. A similar graph that including hemimetabolous insects [41] shows that only 1 other genome
(Schizaphis graminums; Aphidoidea, 1 of 11 hemipteran
genomes) is so C-skewed and A+T% rich. Conversely, the
thysanuran genomes and a subset of Coleoptera are very
A-skewed and distinctly more A+T% balanced (Figure
4A). This pattern is discussed in more detail in the following section.
Codon Usage
PCGs from Strepsiptera and the holometabolan dataset
were imported into INCA v2.1 [42] to analyze codon
usage. M. australiensis and X. vesparum are compared in
Figure 5: codon bias is significantly relaxed in X. vesparum
(2-tailed paired T-Test of ENC/MILC vectors (see methods); P < 1 × 10-5), and an apparent switch in threonine
(T) preference from ACU to ACA has occurred. In Figure
6A, these levels are assessed in context, alongside the
holometabolan dataset using the ENC and MILC measures of codon bias for individual PCGs (see methods;
[43]). In X. vesparum, all 12 PCGs occupy a much more
relaxed, spread apart region of ENC/MILC space (the
higher the value, the less codon bias), with average gene
ENC and MILC values of 36.26 and 0.71 respectively. In
M. australiensis these are 29.63 and 0.56, and in Holometabola, they average 32.77 (ENC) and 0.62 (MILC) over
all genes. It follows that the values of codon bias among
PCGs in M. australiensis should be found in the densest
cluster of background holometabolan PCGs. The thysanuran genomes (empty black circles), alongside three
coleopteran genomes (forming the isolated cluster of
empty blue circles with MILC values > 1) have particularly
balanced codon usage. Although beetles generally follow
this rule; Pyrophorus divergens, Tetraphalerus bruchi and Tribolium castaneum in particular, have unusually high MILC
values. Interestingly, unlike the thysanurans, these do not
result in correspondingly elevated ENC values, possibly
because of differences in the way MILC and ENC estimate
bias [43].
These results are consistent with prevailing neutral mutational theories positing that genomic G+C content is the
most significant factor in determining codon bias
between organisms [44-47]. This may explain why X.
vesparum has significantly relaxed ENC-MILC values,
whose global G+C content is 5% higher than M. australiensis. For the thysanuran and beetle genomes, although
G+C content in the 3rd codon position of individual PCGs
is not necessarily elevated (Figure 6B), it is global G+C%
content that appears to matter: it has been documented
that codon bias can be accurately predicted from inter-
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Graphical
tabolan
4 summary
genomesof nucleotide content across 68 holomeGraphical summary of nucleotide content across 68
holometabolan mt genomes. Measured in bp percentage
(Y-axis) and level of nucleotide skew (X-axis) (described in
methods, following [41]). Green: Diptera; Orange: Lepidoptera; Red: Hymenoptera; Blue: Coleoptera; Turquoise: Neuropterida; Burgundy: Strepsiptera. 1 and 2 refer to M.
australiensis and X. vesparum respectively. A) A+T% vs Askew B) G+C% vs C-skew. 3 genomes from Thysanura (in
black), the host group for M. australiensis are also shown (see
methods).
genic regions [44], and it is unsurprising that these
genomes should also have the highest genome wide G+C
content across the 68 Holometabola dataset (Additional
file 2). It is noticeable that the coleopteran T. bruchi has a
very A-skewed genome (Figure 4A), which could also
partly explain some of the discrepancies appearing
between the different methods.
Secondary structure of ribosomal and transfer RNAs
With the addition of M. australiensis, a comparative revision of strepsipteran mitochondrial rRNA secondary
structure was possible. Figures 7 and 8 are complete models for the rrnL in M. australiensis and X. vesparum respec-
tively. The addition of new comparative evidence (from
M. australiensis and Apis mellifera [48]) since Carapelli et
al. (2006, [28]) enables some improvement over the structural model for X. vesparum. These modifications are highlighted in blue. Regions in which sequence variability is
still too high for good structural covariation are highlighted in red. Overall, M. australiensis predictions are
largely consistent with current insect consensus models
but substantial portions of secondary structure remain
problematic in X. vesparum. For example, the helix at the
base of domain IV in M. australiensis is supported by comparative evidence from Drosophila melanogaster and Apis
mellifera. In X. vesparum, most of this primary sequence in
this helix is highly divergent and covarying base-pairing
cannot be identified. Where this helix terminates in the
single-strand bulge however, the base pairing AUG-UAU
is supported by M. australiensis (AAG-UUU) which provides additional corroborative support for the bps in D.
melanogaster and A. mellifera, which have, AAG-UUU and
AGG-UCU respectively.
rrnS secondary structural models for M. australiensis and
X. vesparum are presented in Figure 9. Absence of sequence
across domain I precludes a complete analysis, but partial
models for domains II and III are generally conserved
across Strepsiptera. Regions highlighted in red are more
labile, and adequate consensus models for these structures are still lacking. The structural predictions for 18
tRNA genes are given in Figure 10. There is less scope for
variation within extremely length-constrained tRNA
genes. Despite this, the TψP stem is still too variable to be
useful in a deep comparative framework: even between M.
australiensis and X. vesparum, base-pairings demonstrate
little covariation. The three remaining stem-loop structures are more conserved across Strepsiptera, and more
useful in a comparative structural context. Within these
regions, X. vesparum demonstrates more substantial modifications to typical insect tRNA structure than M. australiensis - consisting largely of single base or base-pair
substitutions/compensations. For example, the proximal
base-pairs of the acceptor stem of tRNA-Pro (P) in X.
vesparum appears as UCAG-CUGA. In M. australiensis, Lepidoptera (Ochrogaster lunifer [41]) and Hymenoptera
(Vanhornia eucnemidarum [39]) it is CAAA-UUUG. Similarly, the proximal DHU stem consists of a canonical A-U
base-pairing in X. vesparum, where in M. australiensis and
other insect groups it appears as a non-canonical G-U.
Further, the base-pairing C-G, found across disparate
insect lineages is substituted by A-U in the proximal stems
of the tRNA-Lys (K) acceptor and the tRNA-Glu (E) DHU
helices of X. vesparum.
Conclusions
The mitochondrial genome of X. vesparum displays a
number of characteristics that are not shared by its distant
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Codon
Figure usage
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in M. australiensis (red) and X. vesparum (black)
Codon usage in M. australiensis (red) and X. vesparum (black). Codon frequencies are given (out of 1) for individual
amino acids.
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genes. Both strepsipteran genomes are unusually Cskewed and A+T% rich. Interestingly, PCGs are noticeably
truncated in both strepsipteran taxa, but especially so in X.
vesparum which has lost nearly twice as many codons
(154) as M. australiensis (81), equating to 4.3% and 2.2%
fewer codons than the average holometabolan genome
respectively.
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Figure 6 of codon bias across Holometabola
Evaluation
Evaluation of codon bias across Holometabola. A)
Codon bias in M. australiensis (red filled circle) and X.
vesparum (black filled circle) measured using ENC and MILC
indices (described in methods), are compared against remaining Holometabola, whose data points are given as empty circles: the colour scheme follows taxonomic group as in Figure
3 and 4. Data points refer to individual PCGs. B) MILC values
are compared against (G+C)3: which is G+C content at the
3rd codon position of each PCG. This serves as an approximate measure of background nucleotide composition across
individual PCGs.
relative M. australiensis. In the latter, only 2 tRNA translocations disrupt an otherwise ancestral insect genome
organization, whereas in X. vesparum, 3 tRNA translocations and at least 2 duplications have occurred (Figure 2).
Codon bias in X. vesparum is also significantly relaxed: M.
australiensis occupies a much more typical region of ENC/
MILC space (Figures 5 and 6). Further, rRNA secondary
structural model predictions in M. australiensis are much
more consistent with current insect consensus structures
(Figures 7, 8, 9 and 10). In X. vesparum, increased variability at the primary sequence level precludes the assembly of
well corroborated models in several parts of the rRNA
These results provide useful insight into the evolution of
strepsipteran mitochondria spanning the shift from a
largely free-living insect into an extremely unique and
simplified endoparasitoid, since the emergence of Strepsiptera over 100 million years ago [49]. The modifications
to the mitochondrial genomes of X. vesparum and M. australiensis appear consistent with organisms that have
evolved extremely derived lifestyles. M. australiensis represents a transitional phase in the evolutionarily specialization of Strepsiptera, and the composition, architecture
and structure of its genome reflects this. These observations raise important questions about the changing selective environment in (mitochondrial) genomes belonging
to small, and almost entirely host-dependent endoparasitoids. Future investigations into the evolutionary and
developmental origins of this unique biological system
must ensure that strepsipteran species from Mengenillidae
are adequately sampled: wide sampling across this group
is crucial if the underlying ancestral signal is to be maximized.
Methods
Specimen description
DNA was used from a small set of free-flying males collected in light traps, leaving little possibility for contamination through thysanuran host DNA. Nuclear and
mitochondrial gene alignments from a widely sampled
Strepsiptera taxon set (unpublished) confirm this. We
also include 3 thysanuran genomes in Figure 8 to emphasize the genome composition differences between M. australiensis and its target host group. Specimens were
collected on the 16th March 2006 in Australia, Queensland, Blackdown Tablelands NP, South Mimosa Creek, 50
m dstr road, 794 mao. Coordinates as follows: S23
47.687'E 149 04. 195'. Light trap loc 34 (Collectors were
N. Jönsson, T. Malm & D. Williams). Complete species
name: Mengenilla australiensis Kifune & Hirashima 1983.
Genome isolation and sequencing
M. australiensis specimens were extracted for PCR either by
macerating tissue and digesting overnight at 50°C with
Chelex 100 grade resin (Bio-Rad Cat. no 142-1253,
Hemel Hampstead, UK) and enzyme Proteinase K (BIOLINE, Cat. no BIO-37037, London, UK) or by employing
a QIAGEN blood and tissue column extraction protocol.
Amplification of the M. australiensis mitochondrion was
carried out by amplifying three ~3 kb fragments, with
Page 8 of 15
(page number not for citation purposes)
BMC Genomics 2009, 10:603
http://www.biomedcentral.com/1471-2164/10/603
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Figure 7 secondary structure of the rrnL in M. australiensis
Predicted
Predicted secondary structure of the rrnL in M. australiensis. Regions in red receive little comparative support and
remain ambiguous. Canonical A-U and G-C base pairings are given as '-'. G-U, G-A, and other non-canonical bp combinations
are denoted as, '•', 'm' and 'l' respectively. Predicted tertiary interactions are represented by a line. Missing data are coded as
'N'.
intervening sections that did not overlap being amplified
by standard PCR. Long PCR mixes containing 1 μl BIO-XACT long DNA polymerase (BIOLINE, Cat. no. BIO21049), 1 μl dNTPs, 25 μl 2× Polymate additive (BIOLINE, Cat. no. BIO-37041) for A+T rich sequences, 5 μl
10× opti-buffer, 4 μl MgCl2 solution, 3 μl forward/reverse
primers and 5-10 μl of genomic DNA were made up to 50
μl reactions with ultrapure (NANOpure Diamond™)
water. Long PCR cycling parameters were as follows: 92°C
for 2 minutes; 10 cycles of: 92°C for 10 seconds, 53°C for
30 seconds and 68°C for 13 minute; 28 cycles of: 92°C for
15 seconds, 53°C for 30 seconds and 68°C for 14 minute;
with an extension step of 7 minutes at 68°C. Long PCR
fragments were gel extracted (QIAGEN Cat no. 28704,
Hemel Hampstead, UK) and used as templates in subsequent sequencing reactions. Standard PCR mixes contained 0.5 μl Taq polymerase, 1 μl dNTPs, 5 μl MgCl2 free
buffer, 4 μl MgCl2solution, 3 μl forward/reverse primers
and 2 μl of genomic DNA, made up to 50 μl reactions with
ultrapure (NANOpure Diamond™) water. Standard PCR
cycling parameters were as follows: 94°C for 2 minutes,
then 35 cycles of 94°C for 1 minute, T°C for 45 seconds
Page 9 of 15
(page number not for citation purposes)
BMC Genomics 2009, 10:603
http://www.biomedcentral.com/1471-2164/10/603
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Figure
Revised 8secondary structural prediction of the rrnL in X. vesparum
Revised secondary structural prediction of the rrnL in X. vesparum. Regions in red receive little comparative support
and remain ambiguous; regions in blue are structural predictions which have improved with the inclusion of comparative data
from M. australiensis. Structural annotations follow figure 7.
(T varied with primer pair) and 72°C for 1 minute, with
an extension step of 5 minutes at 72°C. For difficult
regions, Phusion high fidelity Taq was utilized, following
the manufacturer's instructions. Standard PCR products
were cleaned using 2 μl of Shrimp Alkaline Phosphatase
(SAP) and 3 μl of Exonuclease I (in a 1:10 SAP buffer dilution) per 50 μl PCR reaction, or fragments were extracted
from agarose gels and sequenced directly. Problematic
fragments that did not sequence directly were inserted
into pGEM-T Easy plasmid vectors (Promega, Southampton, UK), and multiple clones were sequenced for each
fragment. Sequencing reactions were performed using the
BigDye® Terminator v3.1 cycle sequencing kit with the fol-
lowing modifications: 1 μl of BigDye®, 1.5 μl 5× Buffer, 1
μl primer (3.2 pmol), 1-3 μl genomic DNA made up to 10
μl reactions with ultrapure (NANOpure Diamond™)
water. Sequence reads were generated using an Applied
Biosystems 3730xl DNA Analyzer. Sequences were
imported into GridinSoft Notepad (Lite Edition; http://
notepad.gridinsoft.com) and BioEdit (version 7.0.5.3;
[50]) for analysis. Primer pairs employed are given in
Additional file 3. The annotated genome fragment can be
found in Genbank under the accession number
GU188852. Voucher specimens and extractions are
deposited in the Hope Entomological Collections, Oxford
University Museum of Natural History, Oxford, UK, and
Page 10 of 15
(page number not for citation purposes)
BMC Genomics 2009, 10:603
http://www.biomedcentral.com/1471-2164/10/603
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Secondary
9 structural predictions for the rrnS in Strepsiptera
Secondary structural predictions for the rrnS in Strepsiptera. A) M. australiensis, B) X. vesparum. Regions in red receive
little comparative support and remain ambiguous. Structural annotations follow figure 7.
the Swedish Museum of Natural History, Stockholm, Sweden.
Genome charactarization
PCGs and their boundaries were diagnosed via sequence
comparison across alignments derived from the 68
holometabolan genome dataset. Start codons found to be
in-frame and not overlapping with upstream genes on the
same strand were usually identified as 5' gene boundaries.
Stop codons were typically identified as TAA, TAG, TA, or
T. A complete annotation of the M. australiensis genome is
given in Additional file 4. One non-coding fragment, 24
bp in length, was identified between the stop codon of
nad1 and the beginning of tRNA-Ser2. Secondary structural predictions for ribosomal and transfer RNAs were
ascertained by implementing a comparative structural
framework [51-55] (for an online tutorial see the jRNA
web site: http://hymenoptera.tamu.edu/rna/), in conjunction with a thermodynamic-based RNA folding algorithm
(mfold; [56]). mfold gave initial raw estimates of structure:
a tool that is especially useful for variable domains with
highly divergent primary sequence. In this approach,
structures with the lowest thermodynamic stability values
were most highly considered (although structures with
only marginal thermodynamic differences were also eval-
uated) and robust comparative evidence where available,
took priority. The following ribosomal RNA structural
templates from the Comparative RNA Web (CRW) site
database [57] were used: Escherichia coli, Drosophila melanogaster, Drosophila virilis, Chorthippus parallelus and Apis
mellifera [48]. For tRNA genes, the following models were
used as comparative data points: X. vesparum (Strepsiptera: [28]), O. lunifer (Lepidoptera: [41]), V. eucnemidarum (Hymenoptera: [39]) and Cryptopygus antarcticus
(Collembola: [58]), structures were verified for accuracy
in tRNA-Scan [59]. Gene order was assessed across
Holometabola using the inferred ancestral insect arrangement as a comparative standard [29,30]. As a measure of
gene length fluctuation, total and individual gene length
was counted across strepsipteran PCGs, and compared
across the dataset of 68 holometabolan genomes. To
include nad2 gene, the 5' portion (approx. 100 amino
acids) was excluded from analysis so that M. australiensis
could be assessed alongside the entire dataset.
The 68 holometabola mitochondrial genome dataset was
compiled from previously published data across the 6
major holometabolan divisions: Diptera [60-74], Lepidoptera [33,75-81], Hymenoptera [39,82-87], Coleoptera
[88-93], Neuropterida [34,94,95] and Strepsiptera [28].
Page 11 of 15
(page number not for citation purposes)
BMC Genomics 2009, 10:603
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Figure 10 structural predictions for M. australiensis tRNA genes
Secondary
Secondary structural predictions for M. australiensis tRNA genes. Pertinent aspects relating to comparative structural
differences between X. vesparum [28] and other insect lineages [39,41,58] are discussed in the main text.
Nucleotide content and Codon usage
A+T% and G+C% values were calculated for the α strand
of the M. australiensis genome, which runs clockwise in
the direction of transcription from nad2 (Figure 1).
Genomic A- and G-skew measures were calculated using
total nucleotide % values in the following manner: [A-T]/
[A+T] and [G-C]/[G+C] following Perna & Kocher [96]. A
complete list of nucleotide content values for the 68
holometabolan dataset is given in Additional file 3.
Codon usage was investigated by importing the complete
cDNA datasets of 68 available holometabolan mitochondrial genomes, including X. vesparum and the sequenced
genome of M. australiensis into INCA v2.1 [42]. Two independent measures of codon bias: ENC (Effective Number
of Codons used) and MILC (Measure Independent of
Length and Composition) are implemented, whose statis-
Page 12 of 15
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BMC Genomics 2009, 10:603
tical justification and behaviour under simulation differ
markedly [43]. These two independent measures were
combined as a singe vector statistic as follows: √(ENC2 +
MILC2). The shortest gene, atp8 was also excluded from
analyses due to artificially increased ENC/MILC bias in
genes shorter than around 100 amino acids. nad4L was of
borderline length (approx. 90-105 amino acids) and was
retained, although nad4L from Drosophila sechellia and
Melipona bicolor produced arbitrary ENC 'cut-off' values of
61, because ENC behaves less stably at borderline gene
lengths; these data points were removed. Readers are
referred to [43] for a detailed explanation.
Abbreviations
mt: mitochondrial; PCG: protein-coding gene; A+T
region: the putative control region; α strand: leading
strand of transcription; ENC: Effective Number of Codons
used; MILC: Measure Independent of Length and Composition; rrnL and rrnS: large (16S) and small (12S) subunit
ribosomal RNA (rRNA) gene; tRNA genes are denoted as
single letter amino acid IUPAC-IUB abbreviations; atp6
and atp8: ATP synthase subunits 6 and 8; cob: apocytochrome b; cox1-3: cytochrome c oxidase subunits 1-3;
nad1-6 and nad4L: NADH dehydrogenase subunits 1-6
and 4L; MRCA: Most Recent Common Ancestor.
Competing interests
http://www.biomedcentral.com/1471-2164/10/603
Additional file 4
Gene annotation. Detailed summary of genome fragment architecture.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-603-S4.XLS]
Acknowledgements
We owe gratitude to Niklas Jönsson, Tobias Malm and Dawn Williams for
collection of material, to Clive Cook Director of the Queensland Parks and
Wildlife Service - Northern Region for the Collecting Permit (#
WITK03408905), and to B. Viklund (Swedish Museum of Natural History,
Stockholm, Sweden) for donation of material. Thanks must also be
extended to Peter Holland for useful discussions, the Leverhulme Trust
(grant no. F/08 502G) for funding to JK, and the Elizabeth Hannah Jenkinson
fund (University of Oxford) for an award to DPM.
References
1.
2.
3.
4.
5.
6.
The authors declare that they have no competing interests.
7.
Authors' contributions
DPM and AH devised and completed laboratory work.
DPM conducted analyses and early manuscript draughts.
All authors contributed to the final version of the manuscript.
8.
9.
10.
Additional material
Additional file 1
11.
12.
Coding gene lengths. Spreadsheet containing coding gene lengths (in
amino acids) across the Holometabola dataset.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-603-S1.XLS]
13.
Additional file 2
15.
Nucleotide content. spreadsheet of genomic nucleotide content, A-skew
and C-skew across the Holometabola dataset.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-603-S2.XLS]
16.
Additional file 3
19.
Primers. List of primer pairs used in the study.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712164-10-603-S3.XLS]
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